Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Exogenous nitric oxide stimulates cell proliferation via activation of a mitogen-activated protein kinase pathway in ovine fetoplacental artery endothelial cells

Sodium nitroprusside (SNP), a nitric oxide (NO) donor and a nitrovasodilator drug used for patients with hypertensive crisis, has been shown to promote angiogenesis. However, direct evidence showing the involvement of NO in the SNP-induced angiogenesis is not available. Accordingly, we assessed whether NO generated from SNP-stimulated ovine fetoplacental artery endothelial (OFPAE) cell proliferation via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also termed ERK1/2). We observed that SNP dose dependently stimulated (P < 0.05) cell proliferation with a maximal effect at 1 microM and that SNP rapidly (相似文献   

Characterization of protein kinase C-delta in mouse oocytes throughout meiotic maturation and following egg activation

Changes in protein kinase C (PKC) activity influence the progression of meiosis; however, the specific function of the various PKC isoforms in female gametes is not known. In the current study, the protein expression and subcellular distribution profile of PKC-delta (PKC-delta), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-delta, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC-delta distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC-delta became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC-delta is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.     

Thyrotropin stimulates progesterone secretion by luteal cells by activation of the cAMP/protein kinase A signaling system: a potential involvement of protein kinase C

Although the corpus luteum (CL) is not known as a target tissue for thyrotropin (TSH), this hormone increases progesterone production by porcine luteal cells cultured in vitro. In this study we investigated the optimal conditions for TSH-stimulated progesterone secretion as well as the involvement of protein kinase A (PKA) and protein kinase C (PKC) in the mechanism of TSH action on porcine luteal cells. To study the PKA and PKC signaling mechanisms, luteal cells collected from mature CL were incubated with the inhibitor of PKA and potent activators of both kinases: PKA-forskolin and PKC-phorbol ester 12-myriistate-13-acetate (PMA). The PKA inhibitor totally suppressed progesterone production in TSH alone, forskolin alone and in TSH plus forskolin-stimulated luteal cells. Forskolin increased basal (P < 0.05) and TSH-stimulated (P < 0.05) progesterone secretion and cAMP accumulation (P < 0.05). Forskolin and PMA added together to control (non-TSH-treated) luteal cells had an additive effect on progesterone production. In TSH-treated cells, the effect of PMA was statistically significant but did not show an additive effect with forskolin. Further PMA did not affect cAMP accumulation in control and TSH-treated luteal cells. Treatment of control and TSH-treated luteal cells with forskolin and PMA together showed the same increase in cAMP accumulation as with forskolin alone. This is the first demonstration that TSH acts on luteal cell steroidogenesis by activation of the cAMP/PKA second messenger system and also that the PKC signaling pathway may be involved in luteal TSH action on the corpus luteum.     

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.     

Calcitonin gene-related peptide stimulates intracellular cAMP via a protein kinase C-controlled mechanism in human ocular ciliary epithelial cells.

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.     

Gs alpha stimulates transcytosis and apical secretion in MDCK cells through cAMP and protein kinase A

Recent evidence suggests a role for heterotrimeric G proteins in vesicular transport. Cholera toxin, which activates Gs alpha by ADP- ribosylation, has been reported to stimulate both apical secretion (Pimplikar, S.W., and K. Simons. 1993. Nature (Lond.). 352:456-458) and apically directed transcytosis (Bomsel, M., and K.E. Mostov. 1993. J. Biol. Chem. 268:25824-25835) in MDCK cells, via a cAMP-independent mechanism. Here, we demonstrate that apical secretion and apically directed transcytosis are significantly stimulated by agents that elevate cellular cAMP. Forskolin, which activates adenylyl cyclase directly, and 8BrcAMP augment both transport processes in MDCK cells. The increase is not limited to receptor-mediated transport (polymeric Ig receptor), since transcytosis of ricin, a galactose-binding lectin, is similarly stimulated. The effects of elevated cellular cAMP on apical secretion and transcytosis are apparently mediated via protein kinase A (PKA), as they are inhibited by H-89, a selective PKA inhibitor. Experiments employing a 17 degrees C temperature block indicate that cAMP/PKA acts at a late, possibly rate-limiting stage in the transcytotic pathway, after translocation of internalized markers into the apical cytoplasm. However, no significant stimulus of apical recycling was observed in the presence of FSK, suggesting that cAMP/PKA either affects transcytosis at a level proximal to apical early endosomes and/or specifically increases the efficiency by which transcytosing molecules are delivered to the apical plasma membrane. Finally, we overexpressed wild-type Gs alpha and a mutant, Q227L, which constitutively activates adenylyl cyclase, in MDCK cells. Although Q227L increased transcytosis more than wild-type Gs alpha, neither construct was as effective as FSK in stimulating transcytosis, arguing against a significant role of Gs alpha in transcytosis independent of cAMP and PKA.     

Nitric oxide-induced F-actin disassembly is mediated via cGMP, cAMP, and protein kinase A activation in rat mesangial cells.

Glomerular mesangial cells contain actin and myosin, and in analogy to vascular smooth muscle cells, they can contract and relax to regulate the glomerular filtration rate. A key molecule that determines hemodynamic properties is nitric oxide, which is produced by nitric oxide synthase isoenzymes located in individual cells of the kidney. The contractility of mesangial cells is based on the interaction of actin microfilament bundles (F-actin) with myosin. We had the notion that nitric oxide influences the shape change of mesangial cells, so we analyzed the signal transduction involved. Chemically unrelated nitric oxide donors induced F-actin dissolution, which was mediated by cGMP but was unrelated to protein kinase G activation. Actin disassembly was achieved with inhibitors of phosphodiesterase-3 and -4 or forskolin-evoked cAMP generation. We assumed that signal transmission involves activation of protein kinase A, and we went on to attenuate F-actin disassembly by protein kinase A inhibition. In conclusion, we found evidence that nitric oxide triggered F-actin dissolution via cGMP generation, inhibition of cAMP-hydrolyzing phosphodiesterase-3, and subsequent protein kinase A activation.     

Leptin potentiates antiproliferative action of cAMP elevation via protein kinase A down‐regulation in breast cancer cells

Previously, we have shown that leptin potentiates the antiproliferative action of cAMP elevating agents in breast cancer cells and that the protein kinase A (PKA) inhibitor KT‐5720 prevented the antiproliferative effects induced by the leptin plus cAMP elevation. The present experiments were designed to gain a better understanding about the PKA role in the antitumor interaction between leptin and cAMP elevating agents and on the underlying signaling pathways. Here we show that exposure of MDA‐MB‐231 breast cancer cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3. Interestingly, intracellular cAMP elevation upon forskolin pretreatment completely abrogated both ERK1/2 and STAT3 phosphorylation in response to leptin and was accompanied by a consistent CREB phosphorylation. Notably, leptin plus forskolin cotreatments resulted in a strong decrease of both PKA regulatory RIα and catalytic subunits protein levels. Importantly, pretreatment with the PKA inhibitor KT‐5720 blocked the forskolin‐induced CREB phosphorylation and prevented both the inhibition by forskolin of leptin‐induced ERK1/2 and STAT3 phosphorylation and the PKA subunits down‐regulation induced by the combination of leptin and forskolin. Altogether, our results indicate that leptin‐dependent signaling pathways are influenced by cAMP elevation and identify PKA as relevantly involved in the pharmacological antitumor interaction between leptin and cAMP elevating drugs in MDA‐MB‐231 cells. We propose a molecular model by which PKA confers its effects. Potential therapeutic applications by our data will be discussed. J. Cell. Physiol. 225: 801–809, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.     

High glucose stimulates adipogenic and inhibits osteogenic differentiation in MG-63 cells through cAMP/protein kinase A/extracellular signal-regulated kinase pathway

Patients with diabetes tend to have an increased incidence of osteoporosis that may be related to hyperglycemia. In this study, we investigated the effects of high glucose on differentiation of human osteoblastic MG-63 cells and involved intracellular signal transduction pathways. Here, we showed that high glucose suppressed the cell growth, mineralization, and expression of osteogenic markers including Runx2, collagen I, osteocalcin, osteonectin, but inversely promoted expression of adipogenic markers including PPARγ, aP2, resistin, and adipsin. Moreover, high glucose significantly increased the intracellular cAMP level in a time-dependent manner and induced ERK1/2 activation. Meanwhile, supplementation of H89, a specific inhibitor of PKA, and PD98059, a specific inhibitor of MAPK/ERK kinase, reversed the cell growth inhibition, the down-regulation of osteogenic markers and the up-regulation of adipogenic markers as well as the activation of ERK under high glucose. These results indicate that high glucose can increase adipogenic and inhibit osteogenic differentiation by activating cAMP/PKA/ERK pathway in MG-63 cells, thereby providing further insight into the molecular mechanism of diabetic osteoporosis.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

Geranylgeraniol enhances testosterone production via the cAMP/protein kinase A pathway in testis-derived I-10 tumor cells

Testosterone levels in men decrease with age; this decline has been linked to various diseases and can shorten life expectancy. Geranylgeraniol (GGOH) is an isoprenoid found in plants that plays an important role in several biological processes; however, its role in steroidogenesis is unknown. Here, we report that GGOH enhances the production of testosterone and its precursor progesterone in testis-derived I-10 tumor cells. GGOH induced protein kinase A (PKA) activity and increased cAMP levels and was found to regulate cAMP/PKA signaling by activating adenylate cyclase without altering phosphodiesterase activity. GGOH also stimulated mRNA and protein levels of steroidogenic acute regulatory protein, a downstream effector in the cAMP/PKA pathway. These results demonstrate that GGOH enhances steroidogenesis in testis-derived cells by modulating cAMP/PKA signaling. Our findings have potential applications for the development of therapeutics that increase testosterone levels in aging men.     

Tumor necrosis factor-alpha stimulates prostaglandin F2alpha secretion by bovine luteal cells via activation of mitogen-activated protein kinase and phospholipase A2 pathways

It has been well demonstrated that tumor necrosis factor-alpha (TNFalpha) stimulates prostaglandin (PG) F2alpha secretion by bovine corpus luteum (CL) in vitro. The objective of the present study was to clarify the intracellular signaling pathway of TNFalpha to stimulate PGF2alpha production in cultured bovine luteal cells. Bovine luteal cells that were obtained from mid- (days 8-12 after ovulation) CL were incubated with TNFalpha (0.6 nM) and/or various compounds as follows: U-73122 (an inhibitor of phospholipase [PL] C), ACA (an inhibitor of PL-A2), H-89 (an inhibitor of protein kinase [PK] A), calphostin C (an inhibitor of PK-C), L-NAME/L-NORG (inhibitors of nitric oxide synthase), and PD98059 (an inhibitor of mitogen-activated protein kinase [MAPK] kinase). Although U-73122 (0. 1-10 microM), H-89 (0.1-10 microM), calphostin C (0.01-1 microM) and L-NAME/L-NORG (1-100 microM) did not affect TNFalpha-induced PGF2alpha secretion by the cultured cells, ACA (1-100 microM) and PD98059 (0.1-100 microM) inhibited TNFalpha-stimulated PGF2alpha secretion by the cells in a dose-dependent fashion (P < 0.05 or lower). These findings suggest that TNFalpha activates the MAPK and PL-A2 pathways in bovine luteal cells to stimulate PGF2alpha secretion.